Single-stranded (ss) as well as double-stranded (ds) rotavirus RNA were used as templates to synthesize cDNA. The ss RNA was produced in vitro transcription, while the ds RNA was purified from rotavirus particles. Since neither ss nor ds rotavirus RNA are polyadenylated, tailing of the 3' ends was necessary prior to dDNA synthesis in order to create an adequate primer for reverse transcription. After polyadenylation and reverse transcription, we obtained ss cDNA copies of apparently full size from several rotavirus strains, including calf strains (NCDV and UK), human strain Wa, canine strain CU-1 and porcine strain OSU. Synthesis of the second strand was achieved after tailing ss cDNA with oligo dC and priming with oligo dG, a procedure that yields complete copies of ds cDNA, since it avoids the subsequent use of S1 nuclease. Ds cDNA produced in this manner was inserted into plasmid PBR 322 and the hybrid used to transform E. coli HB-101. A majority of 300 NCDN transformants studied by colony hybridization contained NCDV DNA. These positive clones are currently being analyzed to determine size and gene origin of the cDNA inserts. Gene amplification and potential expression of rotavirus in bacteria should allow us to analyze the structure and genomic organization of these viruses.